Understanding the functions of the thousands of uncharacterized genes in sequence databases is among the most significant challenges in biology. The primary goal of this proposal is to define the biochemical and physiological functions of 235 uncharacterized genes in NIAID Category A (Bacillus anthracis and Yersinia pestis) and Category B (Brucella abortus and Coxiella burnetii) priority pathogens. These bacterial pathogens are highly infectious and have had a profound effect on global health both historically and currently. Included in this set are 185 predicted protein coding genes, 59 of which have no evidence of expression. The remaining 50 genes in our set are expressed, or are predicted to be expressed as small, non-coding RNAs (sRNAs). Our state-of-the-art research infrastructure at the Howard Taylor Ricketts Regional Biocontainment Laboratory (HTRL) and Argonne National Laboratory (ANL), and our integrated expertise in biochemistry and microbiology make us an ideal team for this interdisciplinary gene function assignment project. Our proposed research program will 1) yield important data on the function of uncharacterized genes in priority pathogens, 2) define the role of numerous uncharacterized genes in processes tied to bacterial pathogenesis, and 3) will produce useful biological and biochemical reagents for the pathogen research community. RELEVANCE: A full understanding of pathogen biology requires an understanding of how pathogen genes function at a biochemical level. Experiments proposed herein will define the biochemical function of uncharacterized genes implicated in disease processes in four NIAID priority pathogens. Project 1: Functional and Biochemical Characterization of B. abortus Stress Response Genes Project Leader: Sean Crosson DESCRIPTION: The immediate goal of this project is to define the cellular and biochemical functions of 57 uncharacterized open reading frames (ORFs), hypothetical proteins, and small non-coding RNAs in B. abortus. Our preliminary data provide evidence that these genes have a functional role in general stress adaptation and chronic mammalian infection. General stress response (GSR)-dependent transcription in B. abortus is controlled by the sigma factor SigE1. Our preliminary investigation of SigE1 and its upstream signaling partner PhyR have demonstrated that these regulatory proteins are required for adaptation to oxidative and acid stress in vitro and for maintenance of chronic infection in a BALB/c murine model. We have experimentally defined a set of 108 genes that are directly or indirectly regulated by SigE1. Within this regulated gene set are 32 uncharacterized open reading frames (ORFs) and nine small non-coding RNAs. Additionally, we have identified 16 hypothetical genes that are adjacent to or in apparent operons with SigE1-regulated ORFs but for which we have no evidence of expression. We propose to: 1) Test strains in which these genes have been deleted in oxidative and acid stress survival assays, and in cell-based and animal infection assays, 2) elucidate the biochemical functions of these hypothetical proteins and SigE1-regulated ORFs in collaboration with our Biochemical Function Technology Core, and 3) characterize the biochemical function of putative non-coding RNAs under transcriptional control of SigE1. These experiments will provide a comprehensive (in vivo and in vitro) functional understanding of conserved and non-conserved B. abortus genes that are currently uncharacterized. RELEVANCE: Brucella spp.are the causative agents of brucellosis, which is among the most common zooneses globally. Due to their high infectivity, easy aerosolization, and debilitating effects on infected individuals. Brucella spp have been classified as a category B bioterror threat by the U.S. Centers for Disease Control (CDC). Experiments proposed herein will define the biochemical function of uncharacterized genes implicated in regulation of B. abortus stress physiology and in the control of chronic brucellosis disease.